SUMMARY ?The highest global malaria prevalence is in Africa, where the most important vectors are members of the Anopheles gambiae species complex, including the widespread Anopheles coluzzii. Our long-term goal is to dissect the natural genetic differences among mosquitoes that underlie malaria transmission, including differences in malaria susceptibility, ecological adaptation, biting behavior, and insecticide resistance. The objective of this project is to identify the genetic and functional mechanisms of the known major genomic control region for natural A. coluzzii susceptibility to wild P. falciparum. This locus is located in a large region of noncoding DNA on chromosome arm 2L. Frequent alleles in the wild population at this locus strongly influence susceptible or resistant malaria infection outcomes. The central hypothesis is that genetic polymorphism of noncoding regulatory elements explains this locus, and therefore an important fraction of vector genetic variation for P. falciparum infection in nature. The rationale is based on the observation that noncoding genetic polymorphisms control >90% of phenotypic variation in animals, while protein-coding sequence polymorphism contributes little to phenotypic variation. The most important noncoding regulatory elements, enhancers, are responsible for the majority of phenotypic variation. Enhancers are regions ~1 kb in size that modulate target gene expression levels independent of their distance or physical orientation to the targets. Enhancers cannot be predicted by sequence signatures, but require functional assays for detection. In an R21 project, we used a high- throughput functional assay to generate the first comprehensive genome-wide map of enhancers in A. coluzzii, as well as maps of the other noncoding regulatory elements, microRNAs and long noncoding RNAs (miRNAs and lncRNAs). Our specific aims will leverage these new genomic resources to study the influence of natural genetic variation in enhancers and other noncoding regulatory elements for malaria infection outcome: Aim 1) Genetically resolve the major natural locus regulating wild malaria infection; Aim 2) Prioritize the candidate noncoding and coding elements within the locus by testing correlation with infection in a wild mosquito panel; Aim 3) Identify functional mechanisms underlying genetic control of malaria infection. This project is significant because it will determine for the first time the genetic and functional mechanisms underlying the major genomic control region for malaria infection of Anopheles in nature. The proposed project is innovative because the effect of genetic variation of noncoding elements, thought to be the main source of phenotypic variation, has barely been examined in any species, especially in malaria vector mosquitoes, and no mechanism of genetic control over vector competence for Plasmodium in nature is currently known. The results are expected to lead to new malaria control tools rooted in the genome and natural ecology of the vector.